1. Field of the Invention
The invention relates to methods and reagents for screening plants, particularly geraniums, for infections with bacterial pathogens. Specifically, the invention is related to nucleic acid amplification and detection methods for specifically detecting a certain pathovar of a bacterial infectious agent in geraniums. The invention provides a nucleic acid hybridization probe that is specific for Xanthomonas campestris pathovar pelargonii, and paired sets of oligonucleotide primers for polymerase chain reaction (PCR)-catalyzed amplification of DNA fragments comprising the nucleic acid sequences of the invention and specific for Xanthomonas campestris pathovar pelargonii. The invention also provides PCR-based methods for detecting Xanthomonas campestris pathovar pelargonii DNA, and in particular for distinguishing pathovar DNA from the DNA of the host plant. Methods for producing detectably-labeled hybridization probes using PCR chain reaction are also provided.
2. Background of the Invention
Cultivation of flowering plants is an important economic resource in many countries in the both developed and developing world. However, disease and infestation with a variety of bacterial pathogens causes significant economic losses in this industry. This is particularly true for vegetatively-propagated plants, in which infection of the stock plant can result in infection of all the progeny derived from such plants, as well as vertical propagation to other stock plants.
Geraniums (Pelargonium) are one such vegetatively-propagated plant afflicted with bacterial pathogens. The most important pathogen of geraniums worldwide is Xanthomonas campestris pathovar pelargonii (Brown) Dye (Knauss and Tammen, 1967, Phytopathology 57: 1178-1181; Strider, 1985, "Geranium" in Diseases of Floral Crops, vol. 2., (Strider, ed.), Praeger Publishers: New York, pp. 142-156). This bacterial pathogen is the etiologic agent of bacterial blight, a disease that often causes significant losses to commercial geranium propagators.
Following infection of the plant, the bacteria become distributed systemically in the xylem of the stems, petioles and leaves. Affected plants are characterized by the development of wilting and water-soaked leaf spots, but when cultivated under conditions unfavorable for symptom development, infected plants often remain asymptomatic. Symptomless geraniums may be inadvertently distributed by domestic and international growers through cuttings taken from infected stock plants. American and European growers currently employ a costly and time consuming quarantine and certification procedure to identify geranium stock plants infected with X. campestris pathovar pelargonii. However, even with extensive indexing and elaborate quarantine programs, sporadic outbreaks of bacterial blight continue to occur in geraniums.
A variety of methods have been used to detect X. campestris pathovar pelargonii in geranium plants, including pathogenicity, biological recovery, and antibody-based tests.
Lazo et al., 1987, Int. J. Syst. Bacteriol. 37: 214-221 disclosed the use of restriction fragment length polymorphism to distinguish pathovars of X. campestris.
Tuinier, 1989, Plant Diseases 73: 875-878 disclosed the use of serological reagents to detect X. campestris pathovar pelargonii in aqueous extracts of geranium plants.
Benedict et al., 1990, Appl. Environ. Microbiol. 56: 572-574 disclosed pathovar-specific antigens of X. campestris pathovar pelargonii detected with monoclonal antibodies.
Anderson & Nameth, 1990, Phytopathology 80: 357-360 disclosed the development of a polyclonal antibody-based serodiagnostic assay for the detection of X. campestris pathovar pelargonii in geranium plants.
Vauterin et al., 1990, Syst, Appl. Microbiol. 13: 166-176 disclose taxonomic analysis of X. campestris pathovars by protein electrophoretic and DNA hybridization techniques.
Vauterin et al., 1991, Gen. Microbiol. 137: 1766-1787 disclosed grouping of X. campestris pathovars by SDS-PAGE analysis of proteins.
Rasmussen & Reeves, 1992, J. Biotechnol. 25: 203-220 disclose DNA probes for the detection of plant pathogenic bacteria.
Hartung, 1992, Plant Diseases 76: 889-893 disclose plasmid-based hybridization probes for detection and identification of X. campestris pathovar citri.
Seal et al., 1992, Appl. Environ. Microbiol. 58: 3751-3758 disclosed specific DNA probes and oligonucleotide primers for PCR detection of Pseudomonas solanacearum DNA.
More recently, the feasibility of exploiting polymerase PCR amplification to detect laboratory isolates of X. campestris pathovar pelargonii has been described.
Manulis et al., 1992, Phytoparasitica 2: 263 disclosed the use of random amplified polymorphic DNA methods for identifying X. campestris pathovar pelargonii.
Louws et al., Can. J. Bot. 14: 245 disclose genomic fingerprinting of X. campestris pathovar vesicatoria by PCR of repetitive sequences.
Hartung et al., 1993, Appl. Environ. Microbiol. 59: 1143-1148 disclose polymerase chain reaction-based detection assay for X. campestris pathovar citri.
Judd et al., 1993, Appl. Environ. Microbiol. 59: 1702-1708 disclose the use of PCR amplification of repetitive sequences to genetically classify strains of Bradyrhizobium japonicum.
Manulis et al., 1994, Appl. Environ. Microbiol. 60: 4094-4099 disclose the use of PCR methods to detect X. campestris pathovar pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.
Sulzinski et al., 1995, J. Phytopathology 143: 429-433 disclosed fingerprinting of Xanthomonas campestris pathovar pelargonii and related pathovars using random-primed PCR.
The prior art does not describe a rapid, economical, specific and sensitive method, or reagents for implementing such method, for detecting X. campestris pathovar pelargonii in cultivated geraniums. Thus, there remains a need in the art for such methods and reagents.